Abstract
Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an explanation of the high incidence of relapse and fatal outcome of many blood malignancies. In this report we have used Jurkat and Raji cell lines as a model for studies of the NKG2D receptor-ligand system in T-and B cell leukemia/lymphoma. Using real-time quantitative RT-PCR and immunoflow cytometry we show that Jurkat and Raji cells constitutively express mRNA and protein for the stress-inducible NKG2D ligands MICA/B and ULBP1 and 2, and up-regulate the expression in a cell-line specific and stress-specific manner. Furthermore, we revealed by electron microscopy, immunoflow cytometry and western blot that these ligands were expressed and secreted on exosomes, nanometer-sized microvesicles of endosomal origin. Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function. Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response. Taken together, our results might partly explain the clinically observed NK-cell dysfunction in patients suffering from leukemia/lymphoma. The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.
Highlights
Several immune mechanisms participate in protecting the host against cancer
Messenger RNA and protein expression of MICA/B and ULBP 1–3 in Jurkat and Raji cells following stress was assessed by realtime quantitative RT-PCR and immunoflow cytometry
We did not find ULBP3 mRNA expression at steady state or after thermal and oxidative stress. These results are in line with the report by Nuckel et al [24] that cancer cells from chronic B cell leukemia patients lacked ULBP3 mRNA
Summary
Several immune mechanisms participate in protecting the host against cancer In these mechanisms the NKG2D receptor-ligand system plays a key role. The activating NK cell receptor Natural Killer Group 2, member D (NKG2D) and its human ligands, the MIC (MHC class I Chain-related proteins A and B) and ULBP (UL-16 Binding Proteins) 1–6, known as RAET1, comprise a powerful cytotoxic system by which foreign, transformed or infected cells are eliminated from the body [1]. Little is known about the regulation and expression of human NKG2D ligands (NKG2DL) in normal and transformed cells, except that they share the common property of induction by a variety of stresses [8]. ADAMand matrix metalloproteases cleaved soluble NKG2DL are believed to bind to the receptor, down-regulate its surface expression on circulating NK- and T cells and, suppress the NKG2D-dependent pathway of cytotoxicity [9,11]. We and others have shown a novel mechanism for bioactive ‘‘soluble’’ NKG2DL secretion as membrane-bound molecules on the surface of normal- and/or tumor-cell exosomes [12,13,14,15]
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